Systematic efforts led by the ENCODE project are underway to characterize all functional DNA elements in the human genome. A growing amount of data generated by ourselves and others has shown that a large portion of the putative regulatory elements are localized far away from gene coding regions. This observation cannot be explained by a simple linear relationship of locations along the genome, and we are unable to address non-linear interactions between functional DNA elements using current technologies. Our goal is to develop an unbiased, whole genome approach for the identification of chromatin interactions involved in transcriptional regulation and other structural and functional roles in the genome. The principal concept of our approach is to use a specially designed DNA oligonucleotide sequence to link different DNA fragments that are non-linearly related in the genome but brought together in close spatial proximity by protein factors in vivo, and extract paired end ditag sequences from the ligated DNA fragments based on the features of the DNA linker. This is followed by mapping the tags to the reference genome sequence, hence revealing the relationship between the paired DNA fragments. The specific aims of this proposal are: 1. To develop an unbiased, whole genome approach for the characterization of long-range chromatin interactions involved in transcription regulation. We developed a prototype protocol for CIA-PET analysis that can extract ditags from the linker-ligated DNA fragments of non-linearly related DNA interactions in S. pombe cells. We will further optimize this methodology and streamline the entire process of library construction, sequencing, and data analysis. In addition, we will rigorously validate the CIA-PET data using a variety of available low-throughput technologies. 2. To adapt CIA-PET technology to mammalian genomes for the identification of long range chromatin interactions involved in transcription regulation. We plan to further improve the specificity of CIA-PET and increase its capacity to suit the need for whole genome analysis in mammalian genomes. We will validate and demonstrate the utility of CIA-PET method in 3 biological systems: a) Chromatin interactions between hemoglobin genes and regulatory elements in mouse erythroid cells. b) Tertiary networks of transcription regulation mediated by Nanog and Oct4 in mouse embryonic stem cells. c) Estrogen receptor mediated chromatin interactions in human breast cancer cells. This project will provide unprecedented insight into the mechanisms of transcription regulations allowing us to better understand how cancer cells develop and how stem cells retain pluripotency.